Alphavirus Compositions and Methods of Use

ABSTRACT

Embodiments are directed compositions related to Eilat virus and uses thereof. Certain aspects are directed to the detection of non-Eilat entities using a chimeric Eilat alphavirus as a capture agent.

This application is a continuation in part of U.S. patent applicationSer. No. 13/923,527 filed Jun. 21, 2013 (pending); which is acontinuation in part of International Patent Application numberPCT/US2011/066996 filed Dec. 22, 2011 (expired), which is anon-provisional application claiming priority to U.S. Provisional PatentApplication Ser. No. 61/459,989 filed Dec. 22, 2010 (expired). Thisapplication claims priority to and incorporates by reference each of theabove referenced applications in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under N01-AI-30027awarded by the National Institutes of Health/National Institute ofAllergy and Infectious Disease. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

A sequence listing required by 37 CFR 1.821-1.825 is being submittedelectronically with this application. The sequence listing isincorporated herein by reference.

BACKGROUND

Current classification of the genus Alphavirus includes 29 species thatcan be classified into nine complexes based on antigenic and/or geneticsimilarities (Powers et al., J Virol., 2001, 75(21):10118-31). BarmahForest, Ndumu, Middelburg, and Semliki Forest complexes consist ofalmost exclusively Old World viruses whereas Venezuelan equineencephalitis (VEE), Eastern equine encephalitis (EEE), and Trocaracomplexes are comprised of New World viruses (id.). Western equineencephalitis (WEE) complex contains both Old World (Whataroa andSindbis) and New World (Aura) viruses as well as recombinant viruses(WEE, Highland J, Fort Morgan, and Buggy Creek) (Powers et al., JVirol., 2001, 75(21):10118-31; Hahn et al., Proc Natl Acad Sci USA.1988, 85(16):5997-6001; Weaver et al., J. Virol. 1997, 71:613-623). Thelatter viruses are decedents of a recombinant virus that obtainednonstructural and capsid genes from an EEE-like virus and the remaininggenes from a Sindbis-like virus (Hahn et al., Proc Natl Acad Sci USA.1988, 85(16):5997-6001; Weaver et al., J. Virol. 1997, 71:613-623).Lastly, the aquatic alphavirus, salmonid alphavirus (SAV), consists oftwo species, salmon pancreatic disease virus (SPDV) and sleepingsickness virus (SDV) that are distantly related to all otheralphaviruses (Weston et al., J Virol. 2002, 76(12):6155-63).

Most alphaviruses are maintained in natural cycles between arthropodvectors, mainly mosquitoes, and susceptible vertebrate hosts (Straussand Strauss, Microbiol Rev. 1994, 58(3):491-562). Occasionally, thesecycles can spill over into the human and animal populations, and cancause disease. Human infections with Old World viruses such as RossRiver (RRV), chikungunya (CHIKV), and Sindbis (SINV) are characterizedby febrile illness, rash and polyarthritis (id.). In contrast,infections with New World viruses, Venezuelan equine encephalitis(VEEV), Eastern equine encephalitis (EEEV) and Western equineencephalitis (WEEV), can cause fatal encephalitis. The ability ofalphaviruses to infect both invertebrates and vertebrates facilitates abroad host range that enables the viruses to be maintained in ecologicalniches with sporadic outbreaks in humans and animals. As such,alphaviruses have been shown to either naturally or experimentallyinfect many vertebrate and invertebrate hosts. Alphaviruses have beenshown to infect mosquito species encompassing three genera (Aedes sp.,Culex sp., Anopheles sp.) as well as ticks and lice (Griffin.Alphaviruses, In: Fields B N, Knipe D M, Howley P M, editors. Virology.5th edition. New York, N.Y.: Lippincott-Raven; Pages 1023-68; Linthicumet al., J Med Entomol. 1991, 28(3):405-9; La Linn et al., J Virol. 2001,75(9):4103-9). Vertebrate hosts include fish, equids, birds, amphibians,reptiles, rodents, pigs, humans, and non-human primates (Griffin.Alphaviruses, In: Fields B N, Knipe D M, Howley P M, editors. Virology.5th edition. New York, N.Y.: Lippincott-Raven; Pages 1023-68; Burton etal., Science 1966, 154(752):1029-31). Consequently, they can be readilycultured in vitro in many vertebrate and invertebrate cell lines (Wayetal., J Gen Virol. 1976, 30(1):123-30; Sarver and Stollar, Virology 1977,80(2):390-400; Igarashi, J Gen Virol. 1978, 40(3):531-44). Whereasdistantly related fish alphaviruses, which are not known to havearthropod vectors, exhibit a narrow host range that is at leastpartially due to temperature (Weston et al., Virology 1999,256(2):188-95; Villoing et al., J Virol. 2000, 74(1):173-83; Graham etal., J Fish Dis. 2008, 31(11):859-68).

The viral factor(s) that underlie the broad host range of mosquito-bornealphaviruses are poorly understood. Host-restricted viruses may provideinsight into these factor(s) and provide vector delivery platforms forexpression or attenuation in specific hosts. But until the presentinvention, no mosquito-only alphaviruses were known in the genusAlphavirus.

SUMMARY

Described herein is a new alphavirus, Eilat virus (EILV), includingnucleic acid compositions, protein compositions, viral compositions, andmethods of using the same.

Certain embodiments are directed to a recombinant alphavirus expressioncassette comprising an alphavirus nucleic acid segment having a nucleicacid sequence that is at least 95% identical to the nucleic acidsequence of SEQ ID NO:1 or a fragment thereof. In certain aspects theexpression cassette is incorporated into isolated nucleic acids,expression vectors, or plasmids comprising all or part of an EILVnucleic acid sequence (SEQ ID NO:1). In certain aspects the nucleic acidis a recombinant DNA. The EILV nucleic acids can have at least 80, 85,90, 95, 98, 99, or 100% sequence identity to SEQ ID NO:1 or any 10, 20,30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000 consecutive nucleotidesegment thereof, including all values and ranges there between. Incertain aspects, a nucleic acid comprises a nucleotide sequence that isat least 80, 85, 90, 95, 98, 99, or 100% identical to all or a part ofthe non-structural protein coding region of EILV (nucleotides 57 to 7304of SEQ ID NO:1, or any 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600,700, 800, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 consecutivenucleotide segment thereof, including all values and ranges therebetween). In a further aspect, a nucleic acid comprises a nucleotidesequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical toall or a part of the structural protein coding region of EILV(nucleotides 7387 to 11088 of SEQ ID NO:1, or any 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, or 10000 consecutive nucleotide segment thereof,including all values and ranges there between).

In certain aspects, the nucleotide segments specifically bind EILVnucleic acids and distinguish EILV from other alphaviruses under theappropriate conditions. In certain aspects the nucleotide segments aresynthetic oligonucleotides. In a further aspect the oligonucleotide is aDNA oligonucleotide or analog thereof.

The EILV nucleic acids described herein can be in the form of isolatedor recombinant nucleic acids or included in a recombinant alphavirusreplicon, a virus, an alphavirus, a viral particle, an alphavirusparticle, an expression cassette, a host cell, an alphavirus vector, andthe like. In still a further aspect, an alphavirus nucleic acid sequencecan comprise a heterologous nucleic acid segment. In certain aspects,the heterologous nucleic acid segment can encode a therapeutic protein,an antigen, a toxin, or a marker. In certain aspects the heterologousnucleic acid is configured to produce a fusion protein that is expressedon the surface of the chimeric EILV. The fusion protein can be a variantof a EILV envelope protein with a heterologous protein or peptideattached or fused to EILV envelope protein.

Certain aspects are directed to an isolated, recombinant, and/orpurified EILV polypeptide or peptide having at least 85, 90, 95, 98, 99,or 100% amino acid sequence identity to all or part of the amino acidsequence of SEQ ID NO:3 (EILV non-structural polyprotein) or SEQ ID NO:5(EILV structural polyprotein). The term “polyprotein” refers to apolypeptide that is post-translationally cleaved to yield more than onepolypeptide. “Polypeptide” refers to any peptide or protein comprising achain or polymer of amino acids joined to each other by peptide bonds.“Polypeptide” refers to both short chains of 100 amino acids or less,commonly referred to as peptides, and to longer chains, generallyreferred to as proteins. “Polypeptides” may contain amino acids otherthan the 20 gene-encoded amino acids. “Polypeptides” include amino acidsequences modified either by natural processes, such aspost-translational processing, or by chemical modification techniques,which are well known in the art.

In certain aspects, the isolated and/or purified EILV protein has atleast 85, 90, 95, 98, 99, or 100% amino acid sequence identity to all orpart of the amino acid sequence of an EILV non-structural proteinincluding: an EILV nsP1 (amino acids 1 to 543 of SEQ ID NO:3, or anypeptide having 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,or 400 consecutive amino acids of SEQ ID NO:3 including all values andranges there between), an EILV nsP2 (amino acids 544 to 1352 of SEQ IDNO:3, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, or 700 consecutive amino acids of SEQ ID NO:3including all values and ranges there between), an EILV nsP3 (aminoacids 1353 to 1808 of SEQ ID NO:3, or any peptide of 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 consecutive amino acids ofSEQ ID NO:3 including all values and ranges there between) or an EILVnsP4 (amino acids 1809 to 2415 of SEQ ID NO:3, or any peptide of 5, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500consecutive amino acids of SEQ ID NO:3 including all values and rangesthere between).

In certain aspects, the isolated and/or purified EILV protein has atleast 85, 90, 95, 98, 99, or 100% amino acid sequence identity to all orpart of the amino acid sequence of an EILV structural protein including:an EILV C protein (amino acids 1 to 255 of SEQ ID NO:5, or any peptideof 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 consecutiveamino acids of SEQ ID NO:5 including all values and ranges therebetween), an EILV E2 protein (amino acids 319 to 739 of SEQ ID NO:5, orany peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or300 consecutive amino acids of SEQ ID NO:5 including all values andranges there between), an EILV E1 protein (amino acids 795 to 1233 ofSEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, or 300 consecutive amino acids of SEQ ID NO:5 includingall values and ranges there between), an EILV E3 protein (amino acids256 to 318 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, or50 consecutive amino acids of SEQ ID NO:5 including all values andranges there between), or an EILV 6K (amino acids 740 to 794 of SEQ IDNO:5, or any peptide of 5, 10, 15, 20, 30, or 40 consecutive amino acidsof SEQ ID NO:5 including all values and ranges there between). Incertain aspects, an immunogenic composition comprises all or part of 1,2, 3, 4, 5, 6, 7, 8, or 9 EILV proteins. In a further aspect, animmunogenic composition comprises all or part of one or more EILVstructural proteins. In another aspect, an immunogenic compositioncomprises all or part of one or more EILV non-structural proteins. Incertain aspects the EILV E1, E2, and/or E3 is a fusion proteincomprising a heterologous protein or peptide. In certain aspects theheterologous protein or peptide is immunogenic.

Other embodiments are directed to alphaviruses comprising all or part ofthe EILV nucleic acid sequence of SEQ ID NO: 1. In certain aspects thealphavirus is a recombinant alphavirus. Certain embodiments are directedto an alphavirus having a genome comprising (a) an alphavirus nucleicacid segment that is at least 95% identical to a corresponding segmentof SEQ ID NO:1 and (b) a heterologous gene. In certain aspects, thealphavirus is chimeric and comprises certain segments of an EILValphavirus and other segments from a non-EILV alphavirus. A non-EILValphavirus includes, but is not limited to Ross River (RRV), chikungunya(CHIKV), Sindbis (SINV), Venezuelan equine encephalitis (VEEV), Easternequine encephalitis (EEEV), Western equine encephalitis (WEEV), or anaquatic or Salmonid alphavirus. In certain aspects the Eilat chimericvirus is an EILV/CHIKV chimera. In certain aspects the EILV/CHIK chimerahas a nucleotide sequence 90, 95, 98, 99, to 100% identical to SEQ IDNO:134. An EILV/CHIK chimeric alphavirus can have a plasmid structure asillustrated in FIG. 14. In certain embodiments the chimeric alphavirusonly replicates in an arthropod host. Such chimeric alphaviruses cancomprise an EILV nucleic acid with 1, 2, 3, 4, or 5 of the structuralgene regions coding for C, E3, E2, 6K, or E1 proteins substituted with acorresponding non-EILV region or segment.

Still further embodiments are directed to immunogenic compositionscomprising an EILV nucleic acid, EILV polypeptide, EILV virus, oralphavirus comprising all or part of an EILV nucleic acid or all or partof 1, 2, 3, 4, 5, 6, 7, 8, or 9 EILV proteins. Certain aspects aredirected to one or more recombinant EILV nucleic acid, recombinant EILVpolypeptide, recombinant EILV virus, or recombinant alphaviruscomprising all or part of an recombinant EILV nucleic acid or all orpart of 1, 2, 3, 4, 5, 6, 7, 8, or 9 recombinant EILV proteins. Certainembodiments are directed to virus like particle comprising a recombinantnucleic acid described herein.

Pharmaceutical formulations, such as vaccines, of the present inventioncomprise an immunogenic amount of alphavirus or viral particlecomprising alphavirus proteins or antigens, as disclosed herein, incombination with a pharmaceutically acceptable carrier.

Other embodiments are directed to alphaviruses as arthropod expressionsystems. These expression systems include all or part of the EILVnucleic acid sequence of SEQ ID NO:1 and a heterologous gene (e.g., atoxin) for expression in arthropods (e.g., mosquitoes). For example, theheterologous gene can be a gene (e.g., antisense or short interferingnucleotide) that disrupts replication or hinders transmission of anarthropod-borne infectious disease including, but not limited to,alphavirus infections (e.g., Chikungunya), flavivirus infections (e.g.,Dengue), and malaria (Plasmodium). See, e.g., Ito et al. “Transgenicanopheline mosquitoes impaired in transmission of a malaria parasite.”Nature 417: 452-455 (2002); Olson K E et al. “Genetically EngineeredResistance to Dengue-2 Virus Transmission in Mosquitoes.” Science272(5263):884-86 (1996). In still other embodiments, the heterologousgene can be toxic to the arthropod (e.g., a bacterial gene such as fromWolbachia), thus killing or reducing the longevity of the arthropod.See, e.g., Moreira L et al. “A Wolbachia Symbiont in Aedes aegyptiLimits Infection with Dengue, Chikungunya, and Plasmodium.” Cell139:1268-78 (2009).

Certain embodiments are directed to kits for detecting alphavirusinfection and methods of using such kits to detect alphavirus infection.In certain aspects the methods comprise contacting a biological samplefrom a subject with a substrate having one or more capture antigens(chimeric EILV) attached that are capable of capturing a targetpolypeptide (e.g., an anti non-EILV alphavirus antibody) and detecting acaptured target polypeptide. In certain aspects detection is with alabeled probe bound, directly or indirectly, to the captured targetpolypeptide. In certain aspects the capture agent is a chimeric EILVvirus. In a further aspect the chimeric EILV virus is an EILV/CHIKV,EILV/EEEV, EILV/WEEV, EILV/VEEV chimera. The presence of a targetpolypeptide in the biological sample is indicative of a viral infectionof the subject or exposure of the subject to a target virus. In afurther aspect the target polypeptide is anti-virus antibodies. Incertain aspects the labeled probe is detected by fluorescence detection,chemiluminescence detection, or colorimetric detection. In certainembodiments the EILV chimera is immobilized.

In certain aspects the sample is a biological sample. In a furtheraspect the biological sample is a biological fluid such as sputum,blood, urine, cerebral spinal fluid (CSF), and the like. In certainaspect the sample is blood or a blood fraction.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of nucleic acid, e.g., by chemicalsynthesis or by the manipulation of isolated segments of nucleic acidsby genetic engineering techniques.

An “immunogenic amount” is an amount of alphavirus or viral particlescomprising alphavirus antigens sufficient to evoke an immune response ina subject administered an immunogenic composition. An amount of fromabout 10¹ to about 10¹⁰ plaque forming units (pfu) per dose is believedsuitable, depending upon the age and species of the subject beingtreated. Subjects that can be administered immunogenic amounts of acomposition described herein, e.g., EILV virus or host-range restrictedEILV-containing alphavirus chimeras, include but are not limited tohuman, mammal, and other animal subjects (e.g., horse, donkey, mouse,hamster, monkeys), including livestock and laboratory animals.Administration may be by any suitable route, such as intraperitoneal,intravenous, subcutaneous, or intramuscular injection.

The term “alphavirus” has its conventional meaning, and includes thevarious species of alphaviruses, including the newly identified Eilatvirus, as well as Eastern Equine Encephalitis virus (EEE), VenezuelanEquine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixunavirus, Western Equine Encephalitis virus (WEE), Sindbis virus, SouthAfrican Arbovirus No. 86, Semliki Forest virus, Middelburg virus,Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forestvirus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Unavirus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus,Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus,the salmonid alphaviruses and the like.

The term “alphavirus replicon” is used to refer to a nucleic acidmolecule expressing alphavirus nonstructural protein genes such that itcan direct its own replication (amplification).

The term “alphavirus replicon particle” refers to a virion orvirion-like structural complex incorporating an alphavirus replicon.

Alphavirus-permissive cells are cells that, upon transfection with theviral RNA transcript, are capable of producing viral particles.

The term “expression vector” refers to a nucleic acid that is capable ofdirecting the expression of a sequence(s) or gene(s) of interest. Thevector construct can include a 5′ sequence capable of initiatingtranscription of a nucleic acid, e.g., all or part of an alphavirus.Expressed nucleic acid can code for biologically active alphavirusnon-structural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), and analphavirus RNA polymerase recognition sequence. In addition, the vectorconstruct may include a viral junction region. The vector may alsoinclude nucleic acid molecule(s) to allow for production of virus, a 5′promoter that is capable of initiating the synthesis of viral RNA invitro from cDNA, as well as one or more restriction sites, and apolyadenylation sequence. In addition, the constructs may containselectable markers such as Neo, SV2 Neo, hygromycin, phleomycin,histidinol, and DHFR. Furthermore, the constructs can include plasmidsequences for replication in host cells and other functionalities knownin the art. In certain aspects the vector construct is a DNA construct.

“Expression cassette” refers to a recombinantly produced nucleic acidmolecule capable of directing the expression of one or more proteins.The expression cassette includes a promoter capable of directing theexpression of a target protein(s), and a sequence encoding one or moreproteins, e.g., proteins comprising alphavirus structural protein(s) orheterologous nucleic acids in the context of alphavirus. Optionally, theexpression cassette may include transcription termination, splicerecognition, and polyadenylation addition sites. Promoters include theCMV, MMTV, MoMLV, and adenovirus VA1RNA promoters. In addition, theexpression cassette may contain selectable markers such as Neo, SV2 Neo,hygromycin, phleomycin, histidinol, and DHFR.

“Virus-like particle” refers to an infective and/or immunogenicnon-virus particle containing viral nucleic acids and/or proteins. Incertain aspects, viral envelope proteins are included in a lipid bilayeror a viral nucleic acid is encapsulated within a lipid bilayer.

“Antigen” refers to a molecule containing one or more epitopes (eitherlinear, conformational or both) that will stimulate a host's immunesystem to make a humoral and/or cellular antigen-specific response. Theterm is used interchangeably with the term “immunogen.” The term“antigen” denotes both subunit antigens (i.e., antigens that areseparate and discrete from a whole organism with which the antigen isassociated in nature), as well as non-replicative, killed, attenuated,or inactivated bacteria, viruses, fungi, parasites or other microbes.

An “immune response” to an antigen or composition is the development ina subject of a humoral and/or a cellular immune response to an antigenpresent in the composition of interest. A “humoral immune response”refers to an immune response mediated by antibody molecules, while a“cellular immune response” is one mediated by T-lymphocytes and/or otherwhite blood cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See, forexample, Erickson et al., J. Immunol. 151:4189-4199, 1993; Doe et al.,Eur. J. Immunol. 24:2369-2376, 1994. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique) (reviewedby McMichael, A. J. and O'Callaghan, C. A., J. Exp. Med.187(9):1367-1371, 1998; Mcheyzer-Williams, M. G. et al., Immunol. Rev.150:5-21, 1996; Lalvani, A. et al., J. Exp. Med. 186:859-865, 1997).

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIGS. 1A-1B. (A) Diagram of the EILV genome. Amino acid size of eachprotein is provided, as well as the intergenic region, 5′ and 3′UTRnucleotide size. (B) Cloning strategy of full-length Eilat virus cDNAclone. Endonuclease sites within EILV sequence, and pRS2 restrictionsites and sequence provided.

FIGS. 2A-2B. (A) Alignment of putative nsP1 conserved sequence element(CSE) within the genus Alphavirus. Nucleotides identical to EILV aredisplayed with dots. (B) Alignment of putative polyprotein cleavagesites within the genus Alphavirus. Amino acids identical to EILV aredisplayed with dots.

FIGS. 3A-3C. (A) Alignments of putative subgenomic promoter (A) and 3′CSE (B) within the genus Alphavirus. Nucleotides identical to EILV aredisplayed with dots. (C) Phylogenetic tree of representative Alphavirusspecies generated from concatenated nonstructural and structural genesnucleotides by using Bayesian method. Mid-point rooted tree is shownwith posterior probabilities on major branches.

FIG. 4. Alignments of putative E1 fusion peptide and ribosomal bindingsite within the genus Alphavirus. Amino acids identical to EILV aredisplayed with dots.

FIGS. 5A-5B. Phylogenetic trees of representative Alphavirus speciesgenerated from structural (A) and nonstructural (B) gene nucleotides byusing Bayesian method. Mid-point rooted trees are shown with posteriorprobabilities on major branches.

FIGS. 6A-6B. Complement fixation (A) and hemagglutination-inhibition (B)tests with Eilat virus and other alphavirus antigens and hyperimmunemouse ascitic fluids (MIAF). *Reciprocal of heterologoustiter/reciprocal of homologous titer.

FIGS. 7A-7B. In vitro characterization of Eilat virus. EILV plaque size3 days post infection on C7/10 cells in a 6-well plate (A). Synthesis ofvirus-specific RNA in C7/10 cells infected with EILV and SINV, analyzedby agarose gel electrophoresis (B). Lane 1=mock-infected cells, lane2=SINV, lane 3=EILV.

FIGS. 8A-8B. Eilat virus particle morphology by cryo-EM and TEM. EILVvirions embedded in vitreous ice (A). Virions budding from the surfaceof C7/10 cells (B).

FIGS. 9A-9B. Growth kinetics of Eilat virus on representativeinvertebrate (A) and vertebrate (B) cell lines. Monolayers were infectedat MOI of 10. Supernatants were collected at indicated intervalspost-infection and titrated on C7/10 cell monolayers. Each data pointrepresents the average titer of samples taken from triplicateinfections.

FIGS. 10A-10C. Illustrates the infectivity of EILV across various hostcells. (A) Diagram of an EILV marker construct. (B) Results of infectionof insect cell lines with a virus comprising the EILV marker genome,light and fluorescent image. (C) Results of infection of vertebrate celllines with a virus comprising the EILV marker genome, light andfluorescent image.

FIGS. 11A-11B. Illustrates results of electroporation of an EILVencoding ribonucleic acid across various host cells. (A) Diagram of anEILV marker construct. (B) Light image and fluorescent image of variouscell lines four days post electroporation.

FIGS. 12A-12B. Illustrates the infectivity of EILV/SIN structuralconstruct across various host cells. (A) Diagram of an EILV/SIN markerconstruct. (B) Results of infection of cell lines with a viruscomprising the EILV/SIN marker genome, light and fluorescent image.

FIGS. 13A-13B. Illustrates the infectivity of EILV/EEEV structuralconstruct across various host cells. (A) Diagram of an EILV/EEEV markerconstruct. (B) Results of infection of cell lines with a viruscomprising the EILV marker genome, light and fluorescent image.

FIG. 14. Illustration of the plasmid map for an Eilat/Chikungunyachimeric alphavirus.

DESCRIPTION

The genus Alphavirus in the family Togaviridae is comprised of small,spherical, enveloped viruses with a genome consisting of single strand,positive-sense RNA approximately 11-12 kb in length (Kuhn R J.Togaviridae: The viruses and their replication, In: Fields B N, Knipe DM, Howley P M, editors. Virology. 5th edition. New York, N.Y.:Lippincott-Raven; Pages 1001-22). The genome contains two open readingframes: the 5′ two-thirds of the genome encodes four nonstructuralproteins (nsP1, nsP2, nsP3, and nsP4); and the 3′ one-third of thegenome encodes for structural proteins (Capsid, E2, E1). Alphavirusesenter susceptible cells via receptor-mediated endocytosis and replicatein the cytoplasm of infected cells (id.). Following internalization, lowendocytic pH induces a conformational change that exposes E1 fusionpeptide and results in the release of the nucleocapsid (id.).

Since the genome of alphaviruses are capped at the 5′ end and have apoly A tail at the 3′ end, the viral RNA serves as mRNA for translationof nonstructural proteins (id.). The resulting polyprotein issequentially cleaved into four proteins that are responsible for RNAreplication, modification, and proteolytic cleavage (id.).Non-structural proteins facilitate the synthesis of negative andpositive strands as well as the transcription of subgenomic mRNAencoding structural proteins (id.). Following translation, E1 and E2 areprocessed and glycosylated, and E1/E2 heterodimers are inserted into thehost plasma membrane (id.). Capsid proteins interact with one genomicRNA copy to form the nucleocapsid, which interacts with the cytoplasmictail of E2 protein to initiate virion budding from host cell membranesto commence another infectious cycle (id.).

Representative examples of alphaviruses include Aura (ATCC VR-368),Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922),Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equineencephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCCVR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCCVR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg(ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCCVR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus(ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247),Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti(ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis(ATCC VR-69), Venezuelan equine encephalomyelitis virus (ATCC VR-923,ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equineencephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252),Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375), all of which areincorporated herein by reference.

I. EILAT VIRUS

Described herein is a new alphavirus, Eilat virus (EILV), isolated froma pool of Anopheles coustani mosquitoes collected in the Negev desert ofIsrael. Phylogenetic analyses places EILV as a sister to the WesternEquine Encephalitis (WEE) antigenic complex within the main clade ofmosquito-borne alphaviruses. Electron microscopy revealed that, likeother alphaviruses, EILV virions are spherical, roughly 60-70 nm indiameter and bud from the plasma membrane of mosquito cells in culture.EILV readily infects a variety of insect cells with little overtcytopathology. However, in contrast to all other alphaviruses, EILV doesnot infect various mammalian and avian cell lines at 37° C.Evolutionarily, these findings indicate that EILV lost its ability toinfect vertebrate cells. Thus, one use of EILV is in reverse geneticstudies to assess the determinants of alphavirus host range. The EILVgenome (SEQ ID NO:1) includes a 5′ promoter, a non-structural protein(nsPs) coding segment (SEQ ID NO:2), an intergenic region containing asubgenomic promoter (SEQ ID NO:6), a structural protein (sPs) codingregion (SEQ ID NO:4), 3′ promoter, and a poly-A tail.

In one embodiment, the present invention provides an isolated nucleicacid comprising a coding segment having at least 80, 85, 90, 95, 98, 99,or 100% sequence identity to SEQ ID NO:1 or a fragment thereof. A“fragment” can be any 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600,700, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,11000 consecutive nucleotide segment thereof, including all values andranges there between.

In some embodiments, the coding segment comprises a non-structural EILVcoding region, e.g., SEQ ID NO:2, or a fragment thereof. In someembodiments, the coding segment encodes a non-structural EILV protein,or fragment thereof, e.g., nsP1, nsP2, nsP3, and/or nsP4. In oneembodiment, all four of nsP1, nsP2, nsP3, and nsP4 are encoded.

In some embodiments, the coding segment comprises a structural EILVcoding region, e.g., SEQ ID NO:4, or a fragment thereof. In someembodiments, the coding segment encodes a structural EILV protein orfragment thereof, e.g., C, E1, E2, E3, and/or 6K. In some embodiments,the coding segment encodes structural EILV protein C, E1, and/or E2.

In one embodiment, the present invention provides a chimera encoding atleast one EILV protein or fragment thereof and a heterologous gene. Inone embodiment, the chimera encodes at least one structural EILVprotein; in another, it encodes at least one non-structural EILVprotein. The heterologous gene can be, e.g., a therapeutic protein, anantigen, a toxin, or a marker. An antigen can be, e.g., a structuralprotein of another virus, e.g., a non-EILV alphavirus, e.g., VEEV, EEEV,or WEEV. In one embodiment, the heterologous gene encodes C, E1, and/orE2 of a non-EILV alphavirus. In one embodiment, the chimera encodes allthree of C, E1, and E2 of a non-EILV alphavirus. Alternatively, theantigen can be a non-viral antigen. Such viral and non-viral antigensare useful in the manufacture of immunogenic compositions, and inmethods for eliciting immune response in mammals including humans asdiscussed below.

In another embodiment, the heterologous gene is selected for selectiveexpression in arthropods. Particular genes of interest include thosethat disrupt replication or hinder transmission of an arthropod-borneinfectious disease and/or reduce the lifetime of the arthropod.Particularly useful hosts for such host-selective expression systemsinclude mosquitoes (Aedes sp., Culex sp., Anopheles sp.). In oneembodiment, the heterologous gene is expressed in Aedes sp., e.g., Aedesalbopictus or Aedes aegypti.

In certain embodiments, the isolated nucleic acid is incorporated intoan alphavirus vector capable of replicating in arthropods, e.g., Aedessp., but not in mammals, e.g., humans.

II. PHARMACEUTICAL COMPOSITIONS

Certain embodiments are directed to pharmaceutical or immunogeniccompositions comprising an alphavirus nucleic acid, alphavirus vector,alphavirus particle, alphavirus protein, or alphavirus virus, incombination with a pharmaceutically acceptable carrier, diluent,adjuvant, or recipient.

Briefly, the compositions described herein may be formulated in crude orpurified forms. To produce virus in a crude form, virus-producing cellsmay first be cultivated in a bioreactor, wherein viral particles arereleased from the cells into the culture media. Virus may then bepreserved in crude form by adding a formulation buffer to the culturemedia containing the virus to form an aqueous suspension. Within certainembodiments, the formulation buffer is an aqueous solution that containsone or more saccharide, high molecular weight structural additive, andbuffering component in water. The aqueous solution may also contain oneor more amino acids.

The virus or viral particle can be formulated in a purified form. Morespecifically, before adding the formulation buffer, the crude virus orviral particle described above may be clarified by passing it through afilter and then concentrated, e.g., by a cross flow concentrating system(Filtron Technology Corp., Nortborough, Mass.). DNase can be added tothe concentrate to digest exogenous DNA. The digest is then filtered toremove excess media components and to establish the virus or viralparticle in a more desirable buffered solution. The filtrate may then bepassed over an affinity column, e.g., Sephadex S-500 gel column, and apurified virus or viral particle is eluted. A sufficient amount offormulation buffer is then added to this eluate to reach a desired finalconcentration of the constituents and to minimally dilute the virus orviral particle. The aqueous suspension may then be stored, e.g., at −70°C., or immediately dried. The formulation buffer may be an aqueoussolution that contains one or more saccharide, high molecular weightstructural additive, and/or buffering component in water. The aqueoussolution may also contain one or more amino acids.

Crude virus or viral particle may also be purified by ion exchangecolumn chromatography. Briefly, crude virus or viral particles may beclarified by passing it through a filter, followed by loading thefiltrate onto a column containing a highly sulfonated cellulose matrix.The virus or viral particle may then be eluted from the column inpurified form by using a high salt buffer, and the high salt bufferexchanged for a more desirable buffer by passing the eluate over amolecular exclusion column. A sufficient amount of formulation buffer isthen added to the purified virus or viral particle and the aqueoussuspension is either dried immediately or stored, e.g., at −70° C.

The aqueous suspension in crude or purified form can be dried bylyophilization or evaporation at ambient temperature.

In certain aspects, the aqueous solutions used for formulation arecomposed of a saccharide, high molecular weight structural additive, abuffering component, and water. The solution may also include one ormore amino acids. The components act to preserve the activity of thevirus or viral particle upon freezing and lyophilization or dryingthrough evaporation. A saccharide can be lactose, or other saccharides,such as sucrose, mannitol, glucose, trehalose, inositol, fructose,maltose, or galactose. In addition, combinations of saccharides can beused, for example, lactose and mannitol, or sucrose and mannitol.

The high molecular weight structural additive aids in preventing viralaggregation during freezing and provides structural support in thelyophilized or dried state. Within the context of the present invention,structural additives are considered to be of “high molecular weight” ifthey are greater than 5000 m.w. In certain aspects, a high molecularweight structural additive is human serum albumin. However, othersubstances may also be used, such as hydroxyethyl-cellulose,hydroxymethyl-cellulose, dextran, cellulose, gelatin, orpolyvinylpyrrolidone.

The amino acids, if present, function to further preserve viral or viralparticle integrity upon cooling and thawing of the aqueous suspension. Apreferred amino acid is arginine, but other amino acids such as lysine,ornithine, serine, glycine, glutamine, asparagine, glutamic acid oraspartic acid can also be used.

The buffering component maintains a relatively constant pH. A variety ofbuffers may be used, depending on the pH range desired, preferablybetween 7.0 and 7.8. Suitable buffers include phosphate buffer, citratebuffer, and tromethamine.

In certain aspects, a viral or viral particle formulation can contain aneutral salt to adjust the final formulation to an appropriateiso-osmotic salt concentration. Suitable neutral salts include sodiumchloride, potassium chloride or magnesium chloride.

The lyophilized or dehydrated viruses can be reconstituted using avariety of substances, such as water. In certain instances, dilute saltsolutions that bring the final formulation to isotonicity may also beused.

III. ASSAYS USING CHIMERIC ALPHAVIRUSES

The methods described herein may comprise, for example, a method usingchimeric Eilat alphavirus having an affinity and specificity fornon-Eilat antibodies as a reagent. The methods may include, for example,radioimmunoassay, enzyme immunoassay such as ELISA orimmunochromatography assay, coagulation assay (e.g., latex coagulationassay), and/or luminescence immunoassay (e.g., fluorescence immunoassayor chemiluminescence immunoassay). The methods are not limited to aparticular assay method as long as a chimeric Eilat alphavirus describedherein is used.

In certain aspects the method is an enzyme immunoassay such as ELISA orimmunochromatography. Methods can be described by taking an ELISA orimmunochromatography as an example; however, it is understood as amatter of course that the methods are not limited in any respect to theexamples described. In particular, the chimeric Eilat alphavirus can becomplexed with various antibodies in any desired order that results inthe detection of antibodies that bind non-Eilat proteins or viruses.Thus, the methods described herein may include a variety of variantsincluding, for example, direct methods, indirect methods, competitivemethods, non-competitive methods, and sandwich methods. The methods maybe carried out utilizing conventional detection and measurementprinciples well known in the art.

In one example, the methods may comprise a step of immobilizing anantibody (a capture antibody) that binds a chimeric Eilat alphavirus ona support, such as a 96-well plate, beads (e.g., polystyrene beads), atube, or a membrane (e.g., nitrocellulose membrane) and incubating thecaptured chimeric Eilat alphavirus with a sample such as the blood orserum containing an antibody to be detected and/or measured. Thechimeric Eilat alphavirus/target antibody complex is then incubated witha detection reagent, e.g., another reagent that specifically binds thetarget antibody (e.g., a secondary antibody) labeled with an enzyme andforming the enzyme-labeled antibody-chimeric alphavirus conjugate. Thetarget antibody is detected using the detection reagent. These steps maybe carried out by conventional procedures as are well known in the art,with the exception of the chimeric Eilat alphavirus used. For the otherreagents to be used in these steps, conventional reagents may be used.In certain aspects the chimeric Eilat alphavirus/target antibody complexcan be formed prior to capture of the complex on a support or detectionmedium.

One example of a representative enzyme immunoassay, ELISA will bebriefly described below in generic terms. It should be understood,however, that the present invention is not intended to be limited tothis example, and this is illustrated solely for the purpose ofdescribing and example of the ELISA.

Enzyme-linked immunosorbent assays (ELISA) generally comprise binding achimeric Eilat alphavirus with a target antibody to be measured in asample; reacting the antibody bound by the chimeric Eilat alphaviruswith a detection reagent directly or indirectly coupled to a labelforming a detectable conjugate; detecting the conjugate, for examplecontacting the conjugate with a chromogenic substrate to convert thesubstrate into a pigment via the action of the enzyme; and detecting ormeasuring the label to identify the presence of a target antibody in asample.

In certain aspects the chimeric Eilat alphavirus can be directly orindirectly coated or immobilized, for example, on the surfaces of thewells of a 96-well microplate as a support member in order to capture atarget antibody. To the wells of the microplate, a sample or a standardsolution containing the target antibody is then added to form a chimericEilat alphavirus/target antibody conjugate coated or immobilized on thewells of the microplate. In certain aspects the chimeric Eilatalphavirus can be added to the sample first forming thealphavirus/target antibody conjugate that is subsequently captured ordetected.

Next, a detection reagent (e.g., a secondary antibody) labeled with adetectable label is added to the wells and reacted with the conjugatecoated or immobilized thereon to form a detectable conjugate. Thedetection reagent may be chemically labeled with an enzyme to form adetection reagent that specifically binds the conjugate or the targetantibody.

After the formation of a detectable conjugate or complex, a chromogenicsubstrate solution may be added to convert the chromogenic substrateinto a pigment by means of the reaction with the enzyme of theenzyme-labeled secondary antibody. The enzyme reaction is thenterminated with a quenching agent including an acid such as sulfuricacid. After the termination of the enzyme reaction, the target antibodycomplexed with the chimeric Eilat alphavirus is detected or measured bymeasuring the absorbance by means of measurement for the coloration ofthe pigment by the colorimetric method. The amount of target antibody inthe sample may be computed from the absorbance. As described above, itis preferred to carry out the sandwich ELISA using a three-componentsandwich system, that is, an antibody-antigen-antibody sandwich system.

In certain aspects an immunochromatographic assay can be used. Achromatographic support can be used in a kit and/or immunochromatographyassay. An immunochromatographic support can comprise a sample paddisposed at one end of the support, a conjugate pad disposed under thesample pad to allow lateral flow of the sample from the sample pad tothe conjugate pad. Underneath the conjugate pad, a migration zone isdisposed extending in a downstream direction to an absorbent paddisposed at the other end of the support so as for the sample migratedfrom the conjugate pad to laterally flow in and through the migrationmembrane through a test line to a control line. Underneath the migrationzone is a backing sheet supporting the support. Theimmunochromatographic support may be structured in such a manner thatthe sample introduced on the sample pad flows laterally and continuouslyby the capillary action of the structuring materials through theconjugate pad and the migration zone to the absorbent pad.

The sample pad can be made of a material having the physical property oflateral flow, that is, permeating the sample in and through thechromatographic support material by means of capillary action. Thematerial to be used for the sample pad may include, for example,cellulose fibers, glass fibers, polyurethanes, polyacetates, acetatecelluloses, and nylons.

The conjugate pad holds a detection reagent (e.g., a labeled chimericEilat alphavirus or antibody). The conjugate pad can be made ofmaterials including, for example, cellulose fibers, glass fibers ornon-woven cloth. The conjugate pad may be prepared by introducing orimpregnating a given amount of the detection reagent (e.g., a labeledchimeric Eilat alphavirus or antibody) into the conjugate pad and dryingthe material. At the conjugate pad, the target antibody contained in thesample migrates from the sample pad and forms a conjugate with thedetection reagent on or in the conjugate pad, followed by capturing andrecognizing the target antibody in the form of a labeledantibody-antigen complex.

The detection reagent for the conjugate pad may be prepared by coatingor labeling a chimeric Eilat alphavirus with a label, e.g., colloidalmetal particles. Average particle sizes of such colloidal metalparticles may be in the range from approximately 1 to 500 nm, preferablyfrom approximately 1 to 50 nm. The labeling reaction may be performed inaccordance with conventional procedures well known in the art.

The migration zone is a chromatographic carrier and may be in the formof a sheet and can be made of a porous material include, but are notlimited to nitrocellulose membrane, cellulose membrane, nylon membrane,glass fibers, or nonwoven cloth. The materials will allow the migrationof the sample in and through the membrane by capillary action. Themigration zone can be configured to allow the sample in the conjugatepad to lateral flow through the membrane toward the adsorbent pad.

The migration zone can be configured to have a test line positionedbetween the conjugate pad and the adsorbent pad. The test line works asa zone for deciding the presence (positive) or absence (negative) of atarget antibody in a sample. The test line may contain a immobilizingreagent that binds chimeric Eilat alphavirus/target antibody complex. Ifthe labeled chimeric Eilat alphavirus is provided in the conjugate padthen the immobilizing reagent can comprise a reagent having an affinityfor the target antibody. In certain aspects, an Eilat specific antibodyor an antibody that binds the constant region of a target antibody isimmobilized on the membrane.

At the test line of the migration zone, the immobilized complex forms adetectable signal or composition. In certain aspects the immobilizedcomplex is visualized by using particles as a label. By using thecolloidal metal particles or other substances as the label the presenceor absence of the target antibody in the sample is visualized, resultingin realizing precise and simple measurement and an extremely useful toolfor point of care detection or measurement. The migration zone may alsocontain a control line downstream the test line. The control line willcomprise an immobilizing agent specific for the detectably labeledreagent allowing the labeled reagent to be captured and visualized inthe absence of a target antibody.

In one aspects the immunochromatography assay comprises the steps ofintroducing a sample containing an antibody to be measured onto a sampleinlet. The sample migrates to the conjugate pad where the samplecontaining the target antibody flows laterally through the conjugate padand through the migration zone. When entering the conjugate pad anytarget antibody present in the sample is conjugated to a detectionreagent forming a labeled antibody complex. The labeled antibody complexmigrates through the conjugate pad, to and through the migration zone.During migration through the migration zone the complex is brought intocontact with the test line. In the test line, the labeled antibodycomplex is then conjugated with an immobilizing reagent of the testline, forming a three-component conjugate, which is in turn visualizedusing the label. If a target antibody is in the sample, the test line isvisualized and determined to be positive. If the sample does not containdetectable levels of the target antibody the assay would be negative andonly the control line is visualized.

The chimeric alphaviruses described herein can be used to preparevarious compositions or kits for performing the assays described aboveand detecting or diagnosing infection or a pathologic condition. For thepreparation of compositions or kits for diagnosing alphavirus infection,the chimeric alphavirus can be coupled to a substrate. In addition,enzyme-linked immunosorbent assay (ELISA) diagnostic kits comprising thechimeric alphavirus can be used to test samples, and rapidimmunochromatographic diagnostic kits can also be prepared for simpleand easy tests.

Certain embodiments are directed to assays for detection of alphavirusinfection. Chimeras containing EILV replicative machinery (i.e.non-structural proteins, 5′ and 3′ UTRs) and structural proteins of anon-EILV alphavirus are used as a capture agent for detecting non-EILValphavirus antibodies in a sample. Supernatants or concentrated EILVchimeras generated in mosquito cells can be utilized as antigens orreagents in a variety of immunoassay formats, inlcuding ELISAs. The kitsusing chimeric virus described herein can be used to detect targetantibodies such as anti-virus antibodies with minimal cross reactivity.In certain aspects the chimeric Eilat alphavirus can be inactivatedusing irradiation or other means of virus inactivation known in the art.

A sample can be assayed for alphavirus by an antigen-antibody reactionin a conventional immunoassay method. Examples of the immunoassaymethods include radioimmunoassay (RIA), enzyme immunoassay (EIA),enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay(FIA), fluorescence polarization, and immunochromatography.

The capture agent used in one aspect is a chimeric alphavirus that bindsspecifically to non-Eilat alphavirus antibodies in a sample. Dependingon the method, the capture agent may be labeled with a labelingsubstance or may be immobilized on a solid phase. In another aspect asecondary antibody may be labeled and used as a detection reagent.

The label or detection moiety is selected depending on the measurementmethod. For example, when the measurement method is radioimmunoassay(RIA), the labeling substance used is a radioisotope such as ¹²⁵I, ¹⁴Cor ³²P. The label used in enzyme immunoassay (EIA) or enzyme-linkedimmunosorbent assay (ELISA) may be an enzyme such as β-galactosidase,peroxidase, alkali phosphatase, or the like. The label used influorescent immunoassay (FIA) or fluorescence polarization is afluorescent dye such as a fluorescein derivative or a rhodaminederivative. In immunochromatography, an insoluble granular marker or thelike can be used. The various particles can be used for labeling, andparticles may include, but are not limited to latex coloring particlesof organic polymers such as polystyrenes, styrenes, or styrene-butadienecopolymers, or metal particles such as metal colloids, e.g., goldcolloid or silver colloid, or metal sulfides.

The substrate or solid phase for immobilizing the capture agent isappropriately selected depending on the measurement method. The materialof the substrate or solid phase is not particularly limited insofar asit has an affinity for the capture agent. Examples of a substrate orsolid phase include synthetic organic polymer compounds such aspolyvinyl chloride, polyvinylidene fluoride (PVDF), polystyrene, astyrene-divinyl benzene copolymer, a styrene-maleic anhydride copolymer,nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile andpolypropylene, polysaccharides such as a dextran derivative, agarose geland cellulose, and inorganic polymer compounds such as glass, silica geland silicone. These materials maybe those into which functional groupssuch as an amino group, an aminoacyl group, a carboxyl group, an acylgroup, a hydroxyl group and a nitro group have been introduced. Theshape of the substrate or solid phase is also appropriately selecteddepending on the measurement method. For example, the solid phase may beplacoid (e.g., a microtiter plate (ELISA plate) and a disk), particulate(e.g., beads), tubular (e.g., a test tube and a tube), fibrous andmembranous. The method of immobilizing the capture agent on the solidphase may be a method known in the art, such as a physical adsorptionmethod, an ionic bonding method, a covalent bonding method or aninclusion method.

In addition to the capture agent, a kit can contains first and secondantibodies that bind specifically to target. A kit can comprises anumber of capture agent specific for a particular target.

The kit described herein is used to measure alphavirus antibodies in aspecimen by the methods described above. In certain aspects the assaymethods is an enzyme immunoassay method. In a further aspect the assaymethod is an enzyme immunoassay method using a chemiluminescencesubstrate.

At least one antibody contained in the kit is an antibody that bindsspecifically to an antibody produced by the subject from which thesample is obtained.

The kit may further contain a substrate or solid phase for immobilizingthe capture agent. This substrate or solid phase may be the same asdescribed above. When the substrate or solid phase is contained in thekit, the substrate or solid phase may be supplied with the capture agentcoupled to the substrate or solid phase, or the capture agent can besupplied in a separate container.

In addition to those components mentioned above, the kit may furthercontain, for example, a wash for washing wells of a microplate, asubstrate for an enzyme labeling antibodies, and the like. The washincludes a buffer solution containing a salt at a predeterminedconcentration.

In certain aspects a capture agent is contacted with a sample forming acomplex between the capture agent and any target present in the sample.The capture agent/target complex may then be directly detected orcontacted with a detection reagent. In certain aspects the detectionreagent is an antibody that specifically binds antibodies produced bythe subject from which the sample has been obtained. The complex betweenthe capture reagent/target/detection reagent can then be detected ormeasured. When a substrate or solid phase for immobilizing the captureagent, the capture agent in the complex formed in the complex-formingstep has been immobilized on a substrate or solid phase. A detectionreagent (e.g., antibody) in the complex is labeled. Accordingly, thecomplex can be detected by detecting the label of the detection reagentin the detection step.

Examples of a substrate or solid phase include a microtiter plate, aplastic tube, glass beads, etc. The label may be bound directly orindirectly to the detection reagent. For example, the detection reagentis labeled with biotin, then, avidin that binds specifically to biotinis coupled with a label, the detection reagent can be label via abiotin-avidin bonding.

IV. EXAMPLES

The following examples as well as the figures are included todemonstrate preferred embodiments of the invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples or figures represent techniques discovered by theinventors to function well in the practice of the invention, and thuscan be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Eliat Virus, a Newly Identified Host Restricted Alphavirus

A. Results

Virus Isolation. EILV was one of 91 viruses collected during a survey ofthe Negev desert in Israel between 1982-84 (Muriu et al., Malar J. 2008,7:43). Mosquitoes were collected from many parts of the desert includingin the city of Eilat and the isolation was from a pool of Anophelescoustani (Fornadel et al., Vector Borne Zoonotic Dis. 2011,11(8):1173-9). Preliminary characterization showed that the virus wasunable to grow in mammalian cells but could grow to high titers ininsect cells.

Genomic analysis. The sequence of EILV was determined by 454 sequencing.EILV genomic sequence was translated and compared with Sindbis virus todetermine the length of each gene product. A schematic of EILV genome isshown in FIG. 1. The length of untranslated regions (UTRs), intergenicregion, and each gene product is similar to that of other alphaviruses.The coding region nucleotides and deduced amino acids of EILV werecompared with other members within the genus. The nucleotide and aminoacid identity of EILV with other members ranged from 57%-43% and 58% to28%, respectively (Table 1).

Comparison of nucleotide and amino acid identity of structural andnonstructural coding regions of alphaviruses. Upper diagonal displayspercent amino acid identity; lower diagonal contains percent nucleotideidentity. EV TROV AURAV WHATV SINV WEEV EEEV VEEV CHIKV EV 52 43 58 4437 36 37 49 TROV 53 43 57 43 38 38 39 51 AURAV 55 57 47 65 38 39 51 41WHATV 57 56 61 55 39 39 39 53 SINV 56 57 61 70 39 39 52 40 WEEV 52 54 5558 57 70 46 40 EEEV 51 53 53 54 54 64 47 41 VEEV 51 54 54 55 54 58 60 41CHIKV 52 53 53 55 54 53 54 54 RRV 51 53 54 55 55 55 55 54 62 UNAV 52 5454 54 55 53 54 53 62 SFV 53 53 54 55 55 54 55 54 62 MIDV 52 53 54 55 5453 54 54 60 BFV 52 52 53 54 53 53 54 53 56 NDUV 51 52 53 54 53 53 53 5358 SESV 50 51 52 52 51 52 52 52 54 SPDV 43 44 44 44 45 44 45 45 45 RRVUNAV SFV MIDV BFV NDUV SESV SPDV EV 48 39 28 39 38 49 47 28 TROV 52 3928 39 38 51 47 29 AURAV 41 41 30 40 39 41 37 21 WHATV 54 41 31 41 40 5349 30 SINV 41 41 31 41 39 41 37 22 WEEV 40 40 30 40 39 40 38 21 EEEV 4140 30 40 40 40 38 21 VEEV 40 40 30 40 39 40 38 22 CHIKV 66 48 36 46 4258 53 30 RRV 49 38 47 42 60 53 30 UNAV 64 59 46 42 44 39 22 SFV 65 66 3633 33 29 29 MIDV 62 61 63 59 44 39 22 BFV 57 56 58 58 42 39 22 NDUV 5857 59 59 57 53 29 SESV 54 54 54 54 54 54 29 SPDV 46 45 46 45 45 43 43

In both analyses, EILV had the highest identity to Whataroa virus(WHATV) and lowest identity to SPDV. Amino acid comparison of individualprotein was also performed (Table 2). EILV polymerase, nsP4, displayedthe highest amino acid identity with other alphaviruses, whereas nsP3had the least. Overall, EILV proteins shared greater identity with Aura(AURAV), WHATV and SINV than other members. The putative cleavage sitesfor the polyproteins were also compared (FIG. 2B). The nsP4 cleavagesite was the most conserved within the genus even amongst the distantlyrelated aquatic alphaviruses, SESV and SPDV. The cleavage sites of EILVnon-structural and structural proteins had a greater identity withTrocara (TROV), AURAV, WHATV and SINV.

TABLE 2 Comparison of individual EILV proteins within the genusAlphavirus. Percent amino acid identities are shown. Virus nsP1 nsP2nsP3 nsP4 capsid E3 E2 6k E1 Trocara 64 58 30 72 49 41 34 41 46 Aura 7360 36 74 53 46 36 36 47 Whataroa 72 65 36 74 50 42 43 40 49 Sindbis 7165 34 77 53 45 40 45 50 WEE 57 49 29 68 43 44 42 38 49 EEE 56 50 29 6943 42 36 40 47 VEE 56 51 29 68 40 47 34 39 45 Chikungunya 56 53 34 69 4443 36 44 42 Ross River 60 52 30 69 41 45 35 25 42 Una 58 53 32 71 42 4536 28 43 Semliki Forest 58 53 36 69 42 53 35 28 43 Middelburg 59 53 3770 42 46 37 32 42 Barmah Forest 58 52 35 70 45 42 32 34 42 Ndumu 60 5232 70 42 50 33 18 42 SES 53 51 28 65 44 47 30 42 42 SPD 41 38 21 52 3125 24 26 36

The four conserved sequence elements (CSE) were also compared. First CSEis located in the 5′ UTR that serves as a core promoter for RNAsynthesis and is structurally conserved. Utilizing mFold EILV 5′UTRcould form hairpin structures similar to that of SINV (data not shown).The second CSE is a 51 nt sequence within nsP1 gene which likelyfunctions as a replication enhancer. EILV nsP1 CSE shared identity withAURAV, WHATV and SINV (FIG. 2). Similar to 5′ CSE, EILV nsP1 CSE wasable to form similar hairpin structures as SINV (data not shown). Thethird CSE is the 24-nt subgenomic promoter that serves as the promoterfor transcription of the subgenomic RNA. EILV subgenomic CSE sharedsignificant identity with WEEV and EEEV (FIG. 3A). Lastly, the 3′ CSE isa 19-nt element located immediately before the poly-A tail, which servesas the promoter for negative strand synthesis. EILV 3′ CSE was almostidentical to AURAV, EEEV, VEEV and SFV (FIG. 3B).

Lastly, the putative E1 fusion peptide and ribosomal binding site incapsid of EILV were also compared. EILV E1 fusion peptide was identicalto WHATV and shared significant identity with SINV, WEEV, EEEV, VEEV andCHIKV (FIG. 4). Whereas the ribosomal binding site showed greatersequence divergence, with greater identity with AURAV and SINV (FIG. 4).Many of the amino acid differences in the EILV ribosomal binding sitewere present in other viruses.

Phylogenetic analysis. Neighbor joining, maximum likelihood and Bayesianmethods were utilized to determine the relationship of EILV within thealphavirus genus. Trees were generated using full-length, non-structuraland structural nucleotide alignments. The full-length and structuralnucleotide analysis utilizing all three methods placed EILV sister tothe WEE complex (FIG. 5A, FIG. 3C, and data not shown) with highposterior and bootstrap support. The analysis of the non-structuralalignment showed some inconsistency. The neighbor joining method placedEILV sister to WEE complex where as Bayesian and maximum likelihoodanalyses placed EILV within the WEE complex basal to WHATV (FIG. 5B, anddata not shown).

Serological Analysis. Both complement fixation (CF) and hemagglutinationinhibition (HI) tests were also performed to determine the antigenicrelationship of EILV with the genus. In CF test, EILV antigen did notcross react with sera against most members and had minimal crossreactivity with TROV, AURAV, SINV, EEEV, and VEEV (FIG. 6A). In HI test,EILV anti-sera minimally cross-reacted with TROV, SINV, WEEV, and EEEV(FIG. 6B). Purified EILV did not hemagglutinate and EILV anti-serayielded high background with homologous antigen therefore these datawere removed from the analysis.

Rescue of infectious EILV clone, in vitro characterization and TEM EILVcDNA clone was constructed utilizing standard molecular techniques.Virus did not cause any overt cytopathology on C7/10, however at lowercell density EILV infected cells grew at a slower rate (data not shown).EILV formed 3-4 mm plaques 3 days post infection on C7/10 cells (FIG.7A). RNA analysis of EILV infected C7/10 demonstrated that EILV couldproduce similar RNA species as SINV, indicating the synthesis of genomicRNA as well as expression of subgenomic RNA (FIG. 7B). TEM analysis ofEILV virions showed that the virions are spherical in shape, roughly60-70 nm in diameter and bud from the plasma membrane (FIGS. 8A-8B).

In vitro host range. Representative vertebrate (Vero, BHK-21, 293, NIH3T3, and DEF) and invertebrate (C6/36, C7/10, Cu. tarsalis, and P.papatasi) cell lines were used to determine the in vitro host range ofEILV. SINV was used as a positive control as it has been shown to have abroad in vitro host range (Way et al., J Gen Virol. 1976, 30(1):123-30;Sarver and Stollar, Virology. 1977, 80(2):390-400; Igarashi, J GenVirol. 1978, 40(3):531-44).

Both SINV and EILV were able to infect Cu. tarsalis, P. papatasi, C6/36,and C7/10 cells (FIG. 9A and data not shown). EILV grew rapidly to hightiters (>10⁷ pfu/mL) 12-hrs-post infection (hpi) with peak titer rangingfrom 5×10⁷ to 5×10⁸ pfu/mL at 48-hpi. Although both SINV and EILV wereable to infect all four invertebrate cell lines, the infection did notyield any overt cytopathology (data not shown). All vertebrate celllines were readily infected by SINV and showed extensive cytopathologyat 12-hpi (data not shown). Whereas EILV was unable to infect any of thevertebrate cell lines tested and no overt cytopathology was observed(FIG. 9B and data not shown). The initial EILV inoculum decayedsignificantly by 72-hpi and was barely above the limit of detection at96-hpi. These results were also confirmed by infection with EILV-eRFP atMOI of 10 (FIG. 10). EILV infected invertebrate cells expressed eRFP24-hrs-post-infection, whereas no fluorescent signal was observed in anyvertebrate cell lines up to 4-days-post-infection (FIGS. 10B-10C).

Analysis of EILV RNA replication in vertebrate cells. To ascertainwhether the host range restriction was present at the RNA levels,EILV-eRFP clone was in vitro transcribed and electroporated intovertebrate and invertebrate cells. EILV-eRFP was unable to replicate invertebrate cells up to 4 days-post-electroporation, whereas it readilyreplicated in invertebrate cells (FIGS. 11A-11B). This indicates thatthe EILV RNA itself in incapable of replication in vertebrate cells.

Chimeric virus host range. Representative vertebrate and invertebratecell lines were used to determine the in vitro host range of EILV/SINand EILV/EEEV chimeras (FIG. 12 and FIG. 13). The chimeras were EILVbackbones having the structural proteins substituted with sindbis virus(SIN) or eastern equine encephalitis virus (EEEV) structural proteingenes. The chimeric virus maintained the EILV host range, i.e.,arthropod specific replication.

B. Materials and Methods:

Viruses and cells. Eilat and Sindbis (Eg 339) viruses were obtained fromArbovirus Reference Center at the University of Texas Medical Branch.Both viruses were amplified on C7/10 cells and stored at −80° C.

Vero, baby hamster kidney (BHK-21), human embryonic kidney (HEK-293),Duck embryo fibroblast (DEF), mouse fibroblast (NIH 3T3), and Aedesalbopictus (C6/36 and C7/10) cell lines were obtained from the AmericanType Culture Collection. Culex tarsalis and Phlebotomus papatasi cellswere obtained from the Arbovirus Reference Center at the University ofTexas Medical Branch. Cell lines were propagated under conditions of 37°C. or 28° C. and 5% CO₂ in Dulbecco's modified Eagle's medium containing10% fetal bovine serum, sodium pyruvate (1 mM), and Penicillin (100units/mL)-Streptomycin (100 μg/ml). C6/36, C7/10 and Culex tarsalismedia was additionally supplemented with 1% tryptose phosphate brothsolution (Sigma). Phlebotomus papatasi cells were maintained inSchneider's media (Sigma) supplemented with 10% fetal bovine serum andPenicillin (100 units/mL)-Streptomycin (100 μg/ml).

Genomic Sequencing. EILV genome was sequenced by 454 sequencing (RocheDiagnostics Corp.). Briefly, viral RNA was extracted using TRIzol LS(Invitrogen), DNase I (Ambion) treated and cDNA was generated by reversetranscription utilizing Superscript II system (Invitrogen) using randomhexamers linked to an arbitrary 17-mer primer sequence. The cDNA wasRNase H treated and randomly amplified by PCR with random hexamer linked17-mer primer. Products were purified (Qiagen) and ligated to specificadapters for sequencing on the 454 Genome Sequencer FLX (454 LifeSciences) without fragmentation. The removal of primer sequences,redundancy filtering, and sequence assembly was performed by utilizingsoftware programs at the GreenePortal website (available on theWorldWideWeb at tako.cpmc.columbia.edu/Tools/). Sequence gaps werefilled by using primers based on pyrosequencing in both directions withABI PRISM Big Dye Terminator 1.1 Cycle Sequencing kits on ABI PRISM 3700DNA Analyzers (Perkin-Elmer Applied Biosystems). The terminal sequencesfor each virus were amplified using the Clontech Smarter RACE kit(Clontech). Full-length genome was verified by classical dideoxysequencing using primers designed from the draft sequence to createproducts of 1,000 basepairs (bp) with 500 by overlaps.

Cloning and rescue of full-length infectious EILV clone. EILV cDNA clonewas constructed utilizing standard molecular techniques. Briefly, EILVRNA was obtained by infecting C7/10 cells. Viral RNA was isolated fromcell culture supernatant using the QIAamp Viral RNA Mini kit (Qiagen).cDNA was produced by using random hexamers and Superscript III(Invitrogen). cDNA fragments were amplified with Phusion DNA polymerase(New England BioLabs) and EILV primers. Amplified PCR products werepurified using QIAquick Gel Extraction Kit (Qiagen). Fragments werecloned into pRS2 (FIG. 1B). The full-length cDNA clone was confirmed byABI PRISM Big Dye sequencing (Applied Biosystems). EILV infectious clonewas rescued using standard techniques. EILV cDNA was linearized with NotI and in vitro transcribed using SP6 RNA polymerase transcription kit(Ambion). Approximately 4 μg of RNA was electroporated into C7/10 cellsand supernatants were harvested at 48 hrs post-electroporation andstored at −80° C.

Phylogenetic Analysis. Phylogenetic analysis was performed. Alphavirussequences were downloaded from GenBank, aligned in SeaView utilizing theMUSCLE algorithm (Edgar, 2004, Nucleic Acids Research, 32:1792-97; Gouyet al., 2010. Molecular Biology and Evolution, 27:221-224). Thesequences were aligned by deducing the amino acid sequence from openreading frames (ORFs) aligned and then returned to nucleotide sequencesfor subsequent analyses. Sequences were aligned as deduced amino acids(aa) from ORFs and then returned to nucleotide sequences for mostanalyses. The two ORFs were concatenated, and the C-terminus of nsP3 andthe N-terminus of the capsid genes were removed from the analysis asthese sequences display significant sequences divergence and producepoor alignments. Following manual adjustments, the complete alignmentwas split into non-structural and structural protein ORFs. Threeanalyses were performed; neighbor joining (NJ), maximum-likelihood (ML),and Bayesian. NJ analysis was performed utilizing PAUP* version 4.0(Swofford, 1998, PAUP*: phylogenetic analysis using parsimony (*andother methods), version 4. Sunderland, Mass.: Sinauer Associates). Treeswere generated utilizing p-distance algorithm and the robustness of NJphylogeny was evaluated by bootstrap resampling of 1,000 replicates. MLand Bayesian analyses were performed utilizing the PHYLIP package andMetropolis-coupled Markov Chain Monte Carlo (MCMCMC) in MrBayes v3.1.2,respectively (Felsenstein, 1989, Cladistics, 5, 164-166; Ronquist andHuelsenbeck, 2003, Bioinformatics 19, 1572-74). Model test in PAUP wasused to identify the best-fit nucleotide substitution model, GTR+I+Gmodel (Posada and Crandall, 1998, Bioinformatics 14, 817-18). Therobustness of ML and Bayesian phylogeny was evaluated by booststrapresampling of 100 and five million generations, respectively.

Serologic tests. Complement fixation (CF) andHemagglutination-inhibition (HI) tests were performed usingmicrotechniques described previously (Beaty et al., 1989, Arboviruses.Schmidt N J, Emmons R W, eds. Diagnostic Procedures for Viral,Rickettsial and Chlamydial Infections, Sixth Edition, Washington D.C.:American Public Health Association, 797-855). CF was performed using twofull units of guinea pig complement and titers were recorded as thehighest dilutions giving 3 or 4 fixation of complement on a scale of 0to 4. HI tests were performed using immune sera and acetone-extractedascitic fluids. Antigens and anti-sera were obtained from the ArbovirusReference Center at the University of Texas Medical Branch.

Transmission electron microscopy. Thin section and cryo-electron(cryo-EM) microscopy were performed as described previously (Ito andRikihisa, 1981. Techniques for electron microscopy of rickettsiae.Burgdorfer W, Anacker R L, eds. Rickettsiae and Rickettsial Diseases.New York: Academic Press, 213-27; Travassos da Rosa et al., Am J TropMed Hyg. 2001, 64(1-2):93-7; Sherman and Weaver, J Virol., 2010,84(19):9775-82). Briefly, monolayers of C7/10 cells infected at MOI of10, 24 hours-post-infection, cell were fixed in 1.25% formaldehyde, 2.5%glutaraldehyde, 0.03% trinitrophenol and 0.03% CaCl₂ in 0.05 Mcacodylate buffer pH 7.3, and washed in washed in 0.1 M cacodylatebuffer. Cells were scraped, pelleted 1% OsO₄ in 0.1 M cacodylate buffer,en bloc stained with 1% uranyl acetate in 0.1 M maleate buffer pH 5.2,dehydrated in ethanol and embedded in Poly/Bed 812 (Polysciences).Ultrathin sections were cut on Reichert Ultracut S ultramicrotome,stained with 2% aqueous uranyl acetate and 0.4% lead citrate, andexamined in a Philips 201 electron microscope at 60 kV.

For cryo-EM, virus was amplified on C7/10 cells at MOI of 0.5.48-hrs-post-infection supernatants were harvested and clarified bycentrifugation at 2,000 g for 10 min. Virus was precipitated overnightat 4° C. by adding polyethylene glycol and NaCl to 7% and 2.3% (wt/vol)concentrations, respectively. Virus was pelleted by centrifugation at4,000 g for 30 min at 4° C. and precipitate was resuspended in TENbuffer (0.05 M Tris-HCl [pH 7.4], 0.1 M NaCl, 0.001 M EDTA). Virus wasloaded onto a 20-to-70% continuous sucrose (wt/vol) gradient in TENbuffer and centrifuged at 270,000 g for 1 hr. Following centrifugation,visible virus band was harvested using a Pasteur pipette and centrifuged4 times through an Amicon Ultra-4 100-kDa-cutoff filter (Millipore) andresuspened in 1 mL of TEN buffer. The purified virus was applied to theholey films (R2x2 Quantifoil; Micro Tools GmbH; or C-flat; Protochips),blotted with filter paper, and plunged into liquid ethane cooled in aliquid nitrogen bath. Frozen grids were stored under liquid nitrogen andtransferred to a cryo-specimen 626 holder (Gatan, Inc.) under liquidnitrogen before being loaded into a JEOL 2200FS electron microscope,equipped with an in-column energy filter (omega type) and a fieldemission gun (FEG) operating at 200 keV.

RNA analysis. C7/10 monolayers were infected with SINV or EILV at MOI of10, 4 hrs-post-infection cells were be labeled with [³H]uridine (20μCi/ml) in the presence of dactinomycin (ActD) (1 μg/ml) for 3 hrs.Following labeling total cellular RNA was isolated by TRIzol(Invitrogen), denatured with glyoxal in dimethyl sulfoxide and analyzedby agarose gel electrophoresis using previously described conditions(Gorchakov et al., J Virol., 2004 78(1):61-75).

Plaque Assay. Virus titration was performed on freshly confluent C7/10cell monolayers in six-well plates. Duplicate wells were infected with0.1-ml aliquots from serial 10-fold dilutions in growth medium, 0.4 mLof growth media was added to each well to prevent cell desiccation, andvirus was adsorbed for 2 hrs. Following incubation, the virus inoculumwas removed, and cell monolayers were overlaid with 3 mL of overlaycontaining 1:1 mixture of 2% tragacanth and 2× MEM with 5% FBS, 2%tryptose phosphate broth solution, 2% Pen-Strep. Cells were incubated at28° C. in 5% CO₂ for 3 days for plaque development, the overlay wasremoved, and monolayers were fixed with 3 mL of 10% formaldehyde in PBSfor 30 mins. Cells were stained with 2% crystal violet in 30% methanolfor 5 min at RT and excess stain was removed under running water andplaques will be counted.

One-step growth curves. Growth curves were performed on representativevertebrate and invertebrate cell lines in triplicates. Three independentdilution curves of EILV and a single dilution of SINV virus stocks wereperformed to obtain a MOI of 10. Each replicate was used to infect 50%confluent monolayers in 25 cm² flasks. Virus was adsorbed in 1 ml ofgrowth medium for 2 hrs at 37° C. or 29° C. with occasional rocking toprevent cell desiccation. After the inoculum was removed, monolayerswere rinsed five times with 12 ml of PBS to remove unbound virus, and 5ml of growth medium was added to each flask. 0.5-ml aliquot were takenimmediately after as a “time hr 0” (T0) sample and replaced with 0.5 mlof fresh medium. Flasks were placed at 37° C. or 28° C. and furthersamples were taken at 12, 24, 48, 72, and 96 hrs-post-infection. Allsamples were flash frozen in ethanol-dry ice and stored at −80C fortitration.

Infection with EILV-eRFP construct. EILV construct encoding enhanced redfluorescent protein (eRFP) under control of subgenomic promoter wasconstructed utilizing standard cloning techniques. Representativevertebrate (293-HEK, Vero, BHK-21, DEF, NIH 3T3), and invertebrate celllines (C6/36, C7/10 Culex tarsalis and Phlebotomus papatasi) cell lineswere infected at an MOI of 10. Light and fluorescent microscopy imageswere obtained at 24 hour intervals post infection.

Electroporation of EILV-eRFP RNA in vertebrate cells. EILV-eRFP cDNA waslinearized with Not I and in vitro transcribed using SP6 RNA polymerasetranscription kit (Ambion). ≈4 μg of RNA was electroporated intorepresentative vertebrate (293-HEK, Vero, BHK-21, DEF, NIH 3T3), andinvertebrate cell lines (C6/36, C7/10). Light and fluorescent microscopyimages were obtained at 24 hour intervals post infection. SINV-eGFPreplicon was utilized as positive control.

Example 2 Assay for Detecting Alphavirus Infection Using Eilat VirusChimeras

Chimeras containing EILV replicative machinery (i.e. non-structuralproteins, 5′ and 3′ UTRs) and structural proteins of Old (Sindbis) andNew (EEEV) World viruses were generated. Supernatants or concentratedEILV chimeras generated in mosquito cells were utilized as antigens inELISAs. EILV/EEEV chimera diluted 1:100 or 1:1,000 in PBS could bereadily detected by mouse polyclonal anti-sera and produced minimalcross reactivity (Tables 3-5)

A chimera containing EILV replicative machinery and structural proteinsof Chikungunya virus (CHIKV) was generated. Supernatants of mosquitocells, infected with EILV/CHIKV, were diluted 1:2000 in PBS and utilizedas antigen in ELISAs to detect mouse polyclonal anti-sera (Table 6).

TABLE 3 ELISA data generated with EILV/EEEV chimera diluted to 1:100 inPBS. Each data point represents average of 4-5 wells. O.D. (450 nm)Serum Dilution EEEV-antisera VEEV-antisera 1:25 3.45 2.50 1:50 2.93 1.731:100 2.64 1.17 1:200 2.56 1.10 1:400 2.22 0.93 1:800 1.50 0.67 1:16001.42 0.60 1:3200 0.65 0.31 Antibody Dilution O.D. (450 nm) Primaryantibody 1:5,000 0.07 Secondary conjugate 1:5,000 0.06 VSV-anti Gantibody 1:1,000 0.06 Diluent 0.06

TABLE 4 ELISA data generated with EILV/EEEV chimera diluted to 1:1,000in PBS. Primary antibody and secondary conjugate were diluted 1:5,000.Each data point represents average of 3-4 wells. O.D. (450 nm) SerumDilution EEEV-antisera VEEV-antisera CHIKV-antisera 1:40  2.34 1.15 0.151:80  1.76 0.65 0.13 1:160 1.39 0.44 0.10 1:320 0.97 0.37 0.09 1:6400.85 0.29 0.08  1:1280 0.53 0.18 0.07  1:2560 0.34 0.24 0.07  1:51200.22 0.13 0.08 Antibody Dilution O.D. (450 nm) Primary antibody 1:5,0000.06 Secondary conjugate 1:5,000 0.06 VSV-anti G antibody 1:1,000 0.06Diluent 0.06

TABLE 5 ELISA data generated with EILV/EEEV chimera diluted to 1:1,000in PBS. Primary antibody and secondary conjugate were diluted 1:10,000.Each data point represents average of 3-4 wells. O.D. (450 nm) SerumDilution EEEV-antisera VEEV-antisera CHIKV-antisera 1:40  2.30 1.06 0.151:80  1.71 0.60 0.12 1:160 1.80 0.42 0.10 1:320 0.96 0.37 0.09 1:6400.81 0.28 0.08  1:1280 0.52 0.18 0.08  1:2560 0.34 0.25 0.07  1:51200.22 0.13 0.07 Antibody Dilution O.D. (450 nm) Primary antibody 1:10,0000.06 Secondary conjugate 1:10,000 0.06 VSV-anti G antibody 1:1,000  0.07Diluent 0.06

TABLE 6 ELISA data generated with EILV/CHIKV chimera diluted to 1:2,000in PBS. Primary antibody and secondary conjugate were diluted 1:10,000.Each data point represents average of 3-4 wells. CHIKV ELISA O.D. (450nm) Serum Dilution CHIKV-antisera EEEV-antisera 1:50 1.53 0.23 1:1001.40 0.14 1:200 1.19 0.10 1:400 1.08 0.07 1:800 0.94 0.06 1:1600 0.870.05 1:3200 0.79 0.05 1:6400 0.82 0.05 Controls Dilution O.D. (450 nm)Negative (Gamboa) 1:100  0.09 Primary Ab 1:10000 0.05 Secondary Ab1:10000 0.05 Diluent 0.05

ELISA protocol. Antigen was bound to Immulon 2HB flat bottom ELISAplates (Thermo Labsystem). EILV/EEEV chimera was diluted 1:1,000 in PBS(100 μL/well). Plates were incubated overnight 4° C. After incubationthe plates were washed 5 times with 300 μL of 1× PBS containing 0.1%Tween 20. The plates were then blocked with 1× PBS containing 0.1% Tween20 and 3% BSA, incubated for 1 hr at RT or overnight at 4° C., andwashed 5 times with 300 μL of 1× PBS containing 0.1% Tween 20.

The sample was diluted in 1× PBS containing 1% BSA and 0.1% Tween-20.The desired starting dilution were determined. To identify the startingdilutions 2-3 fold serial dilutions were done. Incubated for 1 hr at RTor overnight at 4° C. and washed 5 times with 300 μL of 1× PBScontaining 0.1% Tween 20. 100 μL primary antibody biotin-SP-AffinipureGoat anti-Mouse IgG (Jackson Labs Cat#: 115-065-164) diluted 1:10,000 in1× PBS supplemented with 0.1% Tween-20 and 1% BSA was added. The plateswere incubated for 1 hr at RT or overnight at 4° C. and washed 5 timeswith 300 uL of 1× PBS containing 0.1% Tween 20.

After incubation with primary antibody, 100 μL streptavidin-horseradishperoxidase conjugate (500 units/ml stock, Roche Immunochemical,Indianapolis, IN) diluted 1:10,000 with 1× PBS supplemented with 0.1%Tween-20 and 1% BSA was added. Incubated for 1 hr at RT or overnight at4° C. and washed 5 times with 300 uL of 1× PBS containing 0.1% Tween 20.After addition of conjugate, 100 μL of TMB (3,3′,5,5′-tetramethylbenzidine, Sigma) was added per well. The reaction was stopped by adding100 μL/well of 1N sulfuric acid. The plates were read at 450 nm.

1. A chimeric alphavirus having a genome comprising (a) all or part of anon-structural protein coding segment having at least 95% identity to anon-structural protein coding segment of SEQ ID NO:1 and (b) astructural protein coding segment of chikungunya virus.
 2. The chimericalphavirus of claim 1, wherein the non-structural protein coding segmentof SEQ ID NO:1 encodes nsP1, nsP2, nsP3, and/or nsP4.
 3. The chimericalphavirus of claim 1, wherein the a non-structural protein codingsegment of SEQ ID NO:1 encoding nsP1, nsP2, nsP3, and nsP4.
 4. Thechimeric alphavirus of claim 1, wherein the structural protein codingsegment of the chikungunya virus encodes C, E1, and/or E2.
 5. Thechimeric alphavirus of claim 1, wherein the structural protein codingsegment of the chikungunya virus encodes C, E1, and E2.
 6. Animmunogenic composition comprising the alphavirus of claim
 1. 7. Amethod of stimulating an immune response in a subject comprisingadministering an effective amount of an immunogenic composition of claim6.
 8. A diagnostic kit comprising a chimeric Eilat alphavirus comprisinga heterologous polypeptide of a non-Eilat alphavirus.
 9. The kit ofclaim 8, wherein the non-Eilat alphavirus is selected from the groupconsisting of Ross River (RRV), chikungunya (CHIKV), Sindbis (SINV)Venezuelan equine encephalitis (VEEV), Eastern equine encephalitis(EEEV) and Western equine encephalitis (WEEV) protein.
 10. The kit ofclaim 8, wherein the non-Eilat alphavirus is chikungunya virus.
 11. Thekit of claim 8, wherein the chimeric Eilat alphavirus is coupled to asubstrate.
 12. The kit of claim 11, wherein the chimeric Eilatalphavirus is covalently coupled to a substrate.
 13. The kit of claim 8,further comprising a detection reagent.